1. Field of the Invention
This invention relates to a method for quantitative analysis of a low molecular weight component contained in a protein-containing liquid sample. More particularly, this invention relates to a method for quantitative analysis of low molecular weight components which are contained in a liquid sample originating from body fluids, such as blood, serum, plasma, urine, bone marrow fluid, saliva, and their processed products, and are capable of binding to proteins contained in the liquid sample.
2. Description of Prior Arts
It is well known that quantitative analysis of a low molecular weight component contained in a liquid sample originating from body fluids is sometimes disturbed by a protein contained together in the liquid sample. This is because most of the low molecular weight components are present in the liquid sample in the form of complexes in which said components are bound to the protein, so that the quantitative analysis utilizing physical or chemical characteristics relating to the free low molecular component is disturbed.
Representative examples of the interaction complex between a low molecular weight component and a protein by which the low molecular weight component is easily bound include an interaction complex between thyroid hormone and albumin, thyroxine-binding-globulin (TGB), or prealbumin.
Quantitative analysis of a thyroid hormone such as thyroxine (T.sub.4) present in blood in a trace amount is generally carried out utilizing an antigen-antibody reaction. However, the quantitative analysis of thyroid hormone is ordinarily disturbed because most of the thyroid hormones are present in the liquid sample in the form of complexes in which the thyroid hormones are bound to the proteins such as those described above.
Accordingly, a variety of measures have been employed for keeping the quantitative analysis of a thyroid hormone from disturbance caused by the coexisting protein. Most of measures for eliminating the disturbance caused by the protein utilize addition of an organic solvent to the liquid sample, pH adjustment of the liquid sample, or incorporation of a compound termed "blocker" into the liquid sample. Using one of these measures, the complex between a thyroid hormone and a protein is broken to liberate most of the thyroid hormone, and the so liberated thyroid hormone is then subjected to quantitative analysis.
Among the above-mentioned measures, the incorporation of a blocker has been generally employed because this measure can be easily operated. As the blockers, there are known sodium salicylate, 1-anilino-8-naphthalenesulfonic acid (ANS), and merthiolate (thimerosal).
The incorporation of a blocker into a liquid sample causes separation of the thyroid hormone from the protein under equilibrium hypothetically represented by the following equation. EQU Protein-Thyroid hormone complex+Blocker.revreaction.Protein-Blocker complex+Thyroid hormone
Accordingly, the method employing the blocker is thought to be a method based on the fact that a blocker incorporated into a liquid sample containing protein-thyroid hormone complexes is predominantly bound to the protein, resulting in liberation of the thyroid hormone.
The method employing a blocker has been generally applied to the quantitative analysis of thyroid hormone based on an antigen-antibody reaction. In more detail, said antigen-antibody reaction of thyroid hormone is carried out on a thyroid hormone liberated in the presence of a blocker.
The thyroid hormone is liberated from the protein in the presence of a blocker according to the above-described equilibrium equation. Thus, it is understood that an increase of the rate of reaction utilized for the quantitative analysis of thyroid hormone, resulting in reducing a period of time required for the analysis, as well as improvement on accuracy of the analysis, is accomplished by placing the equilibrium equation to the right, as far as possible. For this reason, the blocker preferably has a high affinity to the protein so that the blocker can be predominantly bound to the protein. The heretofore known blockers such as sodium salicylate, ANS and thimerosal, however, do not seem satisfactory in their affinity to the protein. Particularly, in a liquid sample containing a high concentration of proteins, these known blockers are poor in their blocking activity, that is, to be predominantly bound to the protein to rapidly liberate the low molecular weight components under analysis.
It is naturally assumed that the poor affinity of the known blocker can be compensated for by incorporating the blocker into the liquid sample in a higher concentration. However, an experimental trial has taught that these known blockers are not satisfactorily effective in the blocking activity in a liquid sample containing a high concentration of proteins, even by using a greater amount of the blocker. Particularly, a solution containing a high concentration of ANS shows a colored phase, and such a color in the solution disturbs the colorimetry or fluorometry to be carried out in a latter stage. Also unfavorable is thimerosal, because the thimerosal contains mercury in the molecule and, when an enzymic reaction is involved in the quantitative analysis, the mercury sometimes disturbs the enzymic reaction.